EP0452484B1 - Production de proteines par recombinaison homologue - Google Patents

Production de proteines par recombinaison homologue Download PDF

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EP0452484B1
EP0452484B1 EP91900640A EP91900640A EP0452484B1 EP 0452484 B1 EP0452484 B1 EP 0452484B1 EP 91900640 A EP91900640 A EP 91900640A EP 91900640 A EP91900640 A EP 91900640A EP 0452484 B1 EP0452484 B1 EP 0452484B1
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gene
cells
amplifiable
region
primary
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EP0452484B2 (fr
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EP0452484A1 (fr
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Arthur I. Skoultchi
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Cell Genesys Inc
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
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    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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Definitions

  • the field of this invention is the expression of mammalian proteins.
  • the process of obtaining coding sequences and eliciting their expression has been a long and arduous one.
  • the identification of the coding sequence has frequently involved the construction of libraries, identification of fragments of the open reading frame, examining the flanking sequences, and the like.
  • the coding region has been only small fraction of the total nucleic acid associated with the gene.
  • pseudogenes or multi-membered gene families have obscured the ability to isolate a particular gene of interest. Nevertheless, as techniques have improved, there has been a continuous parade of successful identifications and isolation of genes of interest.
  • expression of mammalian proteins of interest is achieved by employing homologous recombination for integration of an amplifiable gene and other regulatory sequences in proximity to a gene of interest without interruption of the production of a proper transcript.
  • the region comprising the amplifiable gene and the gene of interest may be amplified, the genome fragmented and directly or indirectly transferred to an expression host for expression of the target protein. If not previously amplified, the target region is then amplified, and the cell population screened for cells producing the target protein. Cells which produce the target protein at high and stable levels are expanded and used for expression of the target protein.
  • compositions for production of mammalian proteins of interest in culture.
  • the method employs homologous recombination in a host cell for integrating an amplifiable gene in the vicinity of a target gene, which target gene encodes the protein of interest.
  • the region comprising both the amplifiable gene and target gene will be referred to as the amplifiable region.
  • the resulting transformed primary cells may now be subjected to conditions which select for amplification, or the amplification may be performed subsequently.
  • Transform includes transform, transfect, transduce, conjugation, fusion, electroporation or any other technique for introducing DNA into a viable cell.
  • the chromosomes or DNA of the transformed cells are then used to transfer the amplifiable region into the genome of secondary expression host cells, where the target region, if not previously amplified sufficiently or at all, is further amplified.
  • the resulting cell lines are screened for production of the target protein and secondary cell lines selected for desired levels of production, which cells may be expanded and used for production of the desired protein in culture.
  • the primary cell may be any mammalian cell of interest, particularly mammalian cells which do not grow readily in culture, more particularly primate cells, especially human cells, where the human cells may be normal cells, including embryonic or neoplastic cells, particularly normal cells.
  • Various cell types may be employed as the primary cells, including fibroblasts, particularly diploid skin fibroblasts, lymphocytes, epithelial cells, neurons, endothelial cells, or other somatic cells, or germ cells.
  • skin fibroblasts which can be readily propagated to provide for large numbers of normal cells, embryonic kidney cells, and the like. These cells may or may not be expressing the gene of interest. In those instances where the target gene is inducible or only expressed in certain differentiated cells, one may select cells in which the target gene is expressed, which may require immortalized cells capable of growth in culture.
  • Amplifiable genes include dihydrofolate reductase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • the amplifiable gene will have transcriptional signals which are functional in the secondary or expression host and desirably be functional in the primary host, particularly where amplification is employed in the primary host or the amplifiable gene is used as a marker.
  • the target genes may be any gene of interest, there already having been a large number of proteins of interest identified and isolated with continual additions to the list.
  • Proteins of interest include cytokines, such as interleukins 1-10; growth factors such as EGF, FGF, PDGF, and TGF; somatotropins; growth hormones; colony stimulating factors, such as G-, M-, and GM-CSF; erythropoietin; plasminogen activators, such as tissue and urine; enzymes, such as superoxide dismutase; interferons; T-cell receptors; surface membrane proteins; insulin; lipoproteins; ⁇ 1 -antitrypsin; CD proteins, such as CD3, 4, 8, 19; clotting factors, e.g., Factor VIIIc and von Willebrands factor; anticlotting factors, such as Protein C; atrial naturetic factor, tumor necrosis factor; transport proteins; homing receptors; addressins; regulatory proteins; etc.
  • cytokines
  • constructs will be prepared where the amplifiable gene will be flanked on one or both sides with DNA homologous with the DNA of the target region.
  • the homologous DNA will generally be within 100 kb, usually 50 kb, preferably about 25 kb, of the transcribed region of the target gene, more preferably within 2 kb of the target gene.
  • gene is intended the coding region and those sequences required for transcription of a mature mRNA.
  • the homologous DNA may include the 5'-upstream region comprising any enhancer sequences, transcriptional initiation sequences, the region 5' of these sequences, or the like.
  • the homologous region may include a portion of the coding region, where the coding region may be comprised only of an open reading frame or combination of exons and introns.
  • the homologous region may comprise all or a portion of an intron, where all or a portion of one or more exons may also be present.
  • the homologous region may comprise the 3'-region, so as to comprise all or a portion of the transcription termination region, or the region 3' of this region.
  • the homologous regions may extend over all or a portion of the target gene or be outside the target gene comprising all or a portion of the transcriptional regulatory regions and/or the structural gene. For the most part, the homologous sequence will be joined to the amplifiable gene, proximally or distally.
  • sequence other than the wild-type sequence normally associated with the target gene will be used to separate the homologous sequence from the amplifiable gene on at least one side of the amplifiable gene. Some portion of the sequence may be the 5' or 3' sequence associated with the amplifiable gene, as a result of the manipulations associated with the amplifiable gene.
  • the homologous regions flanking the amplifiable gene need not be identical to the target region, where in vitro mutagenesis is desired. For example, one may wish to change the transcriptional initiation region for the target gene, so that a portion of the homologous region might comprise nucleotides different from the wild-type 5' region of the target gene. Alternatively, one could provide for insertion of a transcriptional initiation region different from the wild-type initiation region between the wild-type initiation region and the structural gene. Similarly, one might wish to introduce various mutations into the structural gene, so that the homologous region would comprise mismatches, resulting in a change in the encoded protein.
  • a signal leader sequence would be introduced in proper reading frame with the target gene to provide for secretion of the target protein expression product.
  • an enhancer in relation to the transcriptional initiation region, which can be provided by, for example, integration of the amplifiable gene associated with the enhancer in a region upstream from the transcriptional initiation regulatory region or in an intron or even downstream from the target gene.
  • flanking sequences will be at least about 150 bp, and may be 12 kb or more, usually not more than about 8 kb.
  • the size of the flanking regions will be determined by the size of the known sequence, the number of sequences in the genome which may have homology to the site for integration, whether mutagenesis is involved and the extent of separation of the regions for mutagenesis, the particular site for integration, or the like.
  • the integrating constructs may be prepared in accordance with conventional ways, where sequences may be synthesized, isolated from natural sources, manipulated, cloned, ligated, subjected to in vitro mutagenesis, primer repair, or the like. At various stages, the joined sequences may be cloned, and analyzed by restriction analysis, sequencing, or the like. Usually the construct will be carried on a cloning vector comprising a replication system functional in a prokaryotic host, e.g., E . coli , and a marker for selection, e.g., biocide resistance, complementation to an auxotrophic host, etc. Other functional sequences may also be present, such as polylinkers, for ease of introduction and excision of the construct or portions thereof, or the like. A large number of cloning vectors are available such as pBR322, the pUC series, etc.
  • the construct may then be used for homologous recombination in the primary cell target.
  • Various techniques may be employed for integrating the construct into the genome of the primary cell without being joined to a replication system functional in the primary host. See for example, U.S. Patent No. 4,319,216, as well as the references cited in the Relevant Literature section.
  • the construct may be inserted into an appropriate vector, usually having a viral replication system, such as SV40, bovine papilloma virus, adenovirus, or the like.
  • the linear DNA sequence vector may also have a selectable marker for identifying transfected cells. Selectable markers include the neo gene, allowing for selection with G418, the herpes tk gene for selection with HAT medium, gpt gene with mycophenolic acid, complementation of an auxotrophic host, etc.
  • the vector may or may not be capable of stable maintenance in the host. Where the vector is capable of stable maintenance, the cells will be screened for homologous integration of the vector into the genome of the host, where various techniques for curing the cells may be employed. Where the vector is not capable of stable maintenance, for example, where a temperature sensitive replication system is employed, one may change the temperature from the permissive temperature to the non-permissive temperature, so that the cells may be cured of the vector. In this case, only those cells having integration of the construct comprising the amplifiable gene and, when present, the selectable marker, will be able to survive selection.
  • selectable marker one may select for the presence of the construct by means of the selectable marker. Where the selectable marker is not present, one may select for the presence of the construct by the amplifiable gene.
  • the amplifiable gene For the neo gene or the herpes tk gene, one could employ a medium for growth of the transformants of about 0.1-1 g/ml of G418 or HAT medium respectively.
  • the selective medium may include from about 0.01-0.25 ⁇ M of methotrexate.
  • the DNA will be introduced into the primary cells.
  • Techniques which may be used include calcium phosphate/DNA co-precipitates, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, polycations, e.g., polybrene, polyornithine, etc., or the like.
  • the DNA may be single or double stranded DNA, linear or circular.
  • Keown et al . Methods in Enzymology (1989), Keown et al ., Methods and Enzymology (1990) Vol. 185, pp. 527-537 and Mansour et al ., Nature , 336 :348-352, (1988).
  • Upstream and/or downstream from the target region construct may be a gene which provides for identification of whether a double crossover has occurred.
  • the herpes simplex virus thymidine kinase gene may be employed since the presence of the thymidine kinase gene may be detected by the use of nucleoside analogs, such as acyclovir or gancyclovir, for their cytotoxic effects on cells that contain a functional HSV-tk gene.
  • nucleoside analogs such as acyclovir or gancyclovir
  • the presence of the marker gene as evidenced by resistance to a biocide or growth in a medium which selects for the presence of the marker gene establishes the presence and integration of the target construct into the host genome. No further selection need be made at this time, since the selection will be made in the secondary expression host, where expression of the amplified target gene may be detected. If one wishes, one can determine whether homologous recombination has occurred by employing PCR and sequencing the resulting amplified DNA sequences. If desired, amplification may be performed at this time by stressing the primary cells with the appropriate amplifying reagent, so that multi-copies of the target gene are obtained. Alternatively, amplification may await transfer to the secondary cell expression host.
  • High molecular weight DNA greater than about 20kb, preferably greater than about 50kb DNA or preferably metaphase chromosomes are prepared from the primary recipient cell strain having the appropriate integration of the amplification vector. Preparation and isolation techniques are described by Nelson and Housman, In Gene Transfer (ed. R. Kucherlapati) Plenum Press, 1986. The DNA may then be introduced in the same manner as described above into the secondary host expression cells, using the same or different techniques than employed for the primary cells.
  • Various mammalian expression hosts are available and may be employed. These hosts include CHO cells, monkey kidney cells, C127 mouse fibroblasts, 3T3 mouse cells, Vero cells, etc. Desirably the hosts will have a negative background for the amplifiable gene or a gene which is substantially less responsive to the amplifying agent.
  • the transformed cells are grown in selective medium containing about 0.01-0.5 ⁇ M methotrexate and, where another marker is present, e.g., the neo gene, the medium may contain from about 0.1-1 mg/ml G418.
  • the resistant colonies are isolated and may then be analyzed for the presence of the construct in juxtaposition to the target gene. This may be as a result of detection of expression of the target gene product, where there will normally be a negative background for the target gene product, use of PCR, Southern hybridization, or the like.
  • the cells containing the construct are then expanded and subjected to selection and amplification with media containing progressively higher concentrations of the amplifying reagent, for example, 0.5-200 ⁇ M of methotrexate for the DHFR gene, and may be analyzed at each selection step for production of the target product.
  • Expansion will include at least duplication and may result in at least 5 copies, preferably 10 copies or more in a tandem relationship.
  • protein production will be increased at least 1.5 fold from expression from a single copy, usually at least 3 fold, preferably at least 5 fold.
  • the various clones may then be screened for optimum stable production of the target product and these clones may then be expanded and used commercially for production in culture. In this manner, high yields of a product may be obtained, without the necessity of isolating the message and doing the various manipulations associated with genetic engineering or isolating the genomic gene, where very large genes can be a major research and development effort.
  • DHFR dihydrofolate reductase
  • CHO Chinese hamster ovary
  • DUKX-Bll cells Urlaub and Chasin, Proc. Natl. Acad. Sci . USA 77 :4216-4220 (1980)
  • secondary recipient are propagated in alpha-medium supplemented with 10% dialyzed fetal bovine serum.
  • the amplification vector is constructed from pUC19 (Yanisch-Perron et al ., Gene 33 :103-119 (1985)).
  • a 1.8 kb Hae II fragment containing a hygromycin B phosphotransferase gene (hph) driven by the herpes simplex virus thymidine kinase (HSV tk) promoter is isolated from pHyg (Sugden et al ., Mol. Cell. Biol . 5 :410-413 (1985)) by digestion with Hae II and gel electrophoresis.
  • Synthetic adaptors are added onto this fragment to convert the Hae II ends into Hin dIII ends and the resulting fragment is joined to pUC19 digested with Hin dIII.
  • the resulting plasmid pUCH contains the hygromycin cassette such that transcription of hph and beta-lactamase are in the opposite orientation.
  • a 1.3 kb Sal I fragment containing a DHFR gene driven by SV40 transcriptional signals is isolated from pTND (Connors et al ., DNA 7 :651-661 (1988)) by digestion with Sal I and gel electrophoresis. This fragment is ligated to pUCH digested with Sal I.
  • the resulting plasmid pUCD contains the DHFR cassette such that DHFR and are transcribed in the same direction.
  • a 1.76 kb Bam HI fragment from the phage F15 (Friezner Degen et al ., J. Biol. Chem . 261 :6972-6985 (1986)) which contains 1.45 kb of DNA flanking the transcriptional start of human tissue plasminogen activator (t-PA) in addition to the first exon and part of the first intron is isolated by gel electrophoresis after Bam HI digestion. This fragment is joined to pUCD following digestion of the latter with Bam HI.
  • the resulting plasmid pUCG has the promoter of the t-PA fragment oriented opposite to that of the DHFR cassette.
  • the t-PA fragment contains a single Nco I site, which is not unique to pUCG.
  • a partial Nco I digest is carried out and a Not I linker is inserted.
  • the resulting plasmid pCG contains a unique Not I site in the t-PA fragment which allows the plasmid to be linearized prior to transformation of the primary human diploid fibroblasts in order to increase the frequency of homologous recombination (Kucherlapati et al ., Proc. Natl. Acad. Sci . USA 81 :3153-3157 (1984)).
  • the plasmid pCG linearized with Not I is introduced into the primary recipients by electroporation employing DNA at 10nM.
  • the resulting cells are then grown in selective medium (EEMEM with 200 ⁇ g/ml hygromycin B). Resistant colonies are isolated and analyzed by PCR (Kim and Smithies, Nucleic Acids Res . 16 :8887-8903 (1988)) using as primers the sequences GCGGCCTCGGCCTCTGCATA and CATCTCCCCTCTGGAGTGGA to distinguish homologous integrants from random ones. Amplification of cellular DNA by PCR using these two primers yields a fragment of 1.9 kb only when DNA from correctly targeted cells is present.
  • Cells comprising the DHFR gene integrated into the t-PA region are expanded and used as a source of genetic material for preparation of secondary recipients.
  • Metaphase chromosomes are prepared Nelson et al ., J. Mol. Appl. Genet . 2 :563-577 (1984)) from recipients demonstrating homologous recombination with the DHFR and are then transformed in DHFR-deficient CHO cells by calcium phosphate mediated gene transfer (Nelson et al ., J. Mol. Appl. Genet . 2 :563-577 (1984)). The cells are then grown in selective medium (alpha-medium containing 200 ⁇ g/mB hygromycin B). Resistant colonies are isolated and analyzed for expression of human t-PA (Kaufman et al ., Mol. Cell. Biol . 5 :1750-1759 (1985)).
  • the cell clones are then grown in selective medium containing progressively higher concentrations of methotrexate (.02-80 ⁇ M, with steps of 4-fold increases in concentration). After this amplification procedure, the cells are harvested and the human t-PA is analyzed employing an ELISA assay with a monoclonal antibody specific for t-PA (Weidle and Buckel, Gene 51 :31-41 (1987)). Clones providing for high levels of expression of t-PA are stored for subsequent use.
  • a clone was obtained by screening a human placental DNA genomic library (Clontech) in EMBL 3-SP6/T7 using two 36 bp oligonucleotide probes 5'-CTGGGTTGCTGAGTTCCGCAAAGTAGCTGGGTCTGG-3' and 5'-CGGGGGTCGGGGCTGTTATCTGCATGTGTGCGTGCG-3' to the presumed promoter region of human erythropoietin. From this clone two subclones were created in pSP72 (Krieg and Melton (1987) Meth. Enzymol.
  • a plasmid pCG.1 was constructed by replacement of the polylinker of pBluescript SK(-) (Stratagene) between the SacI and KpnI sites with a synthetic double stranded 72 base pair DNA fragment (FIG. 1). Referring to FIG. 2, into pCG.1 was cloned between the HindIII and XbaI sites a 678 bp fragment containing the enhancer and promoter of the immediate early gene of human cytomegalovirus (CMV, Boshart et al (1985) Cell 41 , 521-530) obtained by a PCR amplification of the plasmid pUCH.CMV (gift of M.
  • CMV human cytomegalovirus
  • the 620 bp BstEII-XbaI fragment from the pTD.2 was joined by the use of a BstEII-XbaI adapter to pCG.CMV restricted with XbaI to create the plasmid pCG.CMV/EPO, in which the BstEII site of the EPO fragment is next to the promoter end of the CMV fragment.
  • pCG.CMV/EPO was cloned successively a 1.94 kb fragment encoding methotrexate resistance from the plasmid pSV2dhfr (Subramani et al (1981) Mol. Cell. Biol.
  • neo gene was obtained as an XhoI-SalI fragment and the dhfr gene was obtained by PCR amplification using the primers 5'-GGACGCGTGGATCCAGACATGATAAGATA-3' and 5'-GGACGCGTCAGCTGTGGAATGTGTGTCAG-3' designed to add MluI sites at the ends of the resultant fragment.
  • neo and dhfr genes were cloned into the XhoI and MluI sites respectively of pCG.CMV/EPO to give the plasmids pCG.CMV/EPO/DHFR and pCG.CMV/EPO/Neo/DHFR such that their transcription is in the same orientation as that of CMV.
  • the 5 kb BamHI-HindIII fragment from pTD.1 was added via ClaI adapters at the ClaI site of pCG.CMV/EPO/Neo/DHFR to give pCG.HR1. In pCG.HR1, the 5' 5kb EPO fragment is in the same orientation as that of the 620 bp BstEII-XbaI fragment with respect to the original lambda clone.
  • a 9.54 kb fragment containing the 5' 5kb BamHI-HindIII EPO fragment, the dhfr and G418 markers, the CMV enhancer/promoter and the 620 bp BstEII-XbaI EPO fragment can be released from pCG.HRl as a NotI or SacII fragment.
  • This NotI fragment can be used for homologous recombination as it is designed to serve as an omega structure in recombination having 5 kb and 620 bp of homology to facilitate the event (FIG. 3).
  • the DNA was first cut with NotI, then extracted with phenol/chloroform and precipitated by the addition of ethanol before centrifugation.
  • the resultant DNA pellet was resuspended at a concentration of 2 mg/ml in a volume (10 ⁇ l) of 10 mM Tris-HCl, 1 mM EDTA (TE).
  • Transformed primary human 293 embryonal kidney cells (ATCC CRL 1573) were cultured in Cellgro DMEM H16 (Mediatech) supplemented with 10% calf serum, glutamine (2 mM) and penicillin (100 U/ml)/streptomycin (0.1 mg/ml) and grown at 37°C in 5% CO 2 . At 90% confluency, cells were prepared for electroporation by trypsinization, concentration by brief centrifugation and resuspension in PBS at 10 7 cells/0.8 ml. The cells were equilibrated at 4°C, and DNA (50 ⁇ g) restricted with NotI (as described above) was added.
  • the mixture was electroporated at 960 ⁇ F and 260 V with a BioRad Gene Pulser and then iced again for 10 min before plating onto a 10 cm dish. After incubation at 37°C for 48 hr, the cells from a 10 cm dish were split equally among 5 24-well plates in media containig G418 at 0.6 mg/ml (effective concentration). Under these electroporation conditions, 4-10 colonies/well survive drug selection after 2 weeks.
  • pools of clones from the electroporated 293 cells from 4 wells each (representing about 16 colonies) were generated by trypsinizing wells and using 90% of each well for the pool. The remaining 10% of each well was then reseeded back into the well. Genomic DNA was then prepared from each pool as follows. The cells in each pool were pelleted by centrifugation for 2 min.
  • the reaction (50 ⁇ l) contained 10 mM Tris-HCl (pH 9.0 at 25°C), 50 mM KCl, 1.5 mM MgCl 2 , 0.01% gelatin, 0.1% Triton X-100, 200 ⁇ M dNTPs, 1 ⁇ M each of the primers 5'-AAGCAGAGCTCGTTTAGTGAACCG-3' and 5'-TGAGCGTGAGTTCTGTGGAATGTG-3', and 1.5 U of Tag DNA polymerase (Promega).
  • metaphase chromosomes are prepared from the recipients demonstrating homologous recombination with DHFR and transformed in DHFR deficient CHO cells. After isolating resistant colonies and analyzing for expression of EPO, the cell clones are grown in selective medium containing progressively higher concentrations of methotrexate (.02-80 ⁇ M) with steps of 4-fold increases in concentration. The cells are then harvested, cloned and screened for production of EPO. Clones providing for at least 2-fold enhancement of EPO production are isolated.
  • the subject method provides for a novel approach to expression of a wide variety of mammalian genes of interest.
  • the method is simple, only requires the knowledge of a sequence of about 300 bp or more in the region of a target gene, and one may then use substantially conventional techniques for transferring the amplifiable region to an expression host, and production of the desired in high yield.

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Claims (13)

  1. Méthode de production de protéines mammaliennes comprenant :
    la croissance de cellules hôtes mammaliennes d'expression secondaire comprenant des copies multiples d'une région amplifiable comprenant un gène cible hétérologue audit hôte d'expression secondaire et exprimant une protéine d'intérêt et un gène amplifiable, ainsi ledit gène cible est exprimé et ladite protéine est produite ;
    où lesdites cellules d'expression de l'hôte secondaire sont produites par la méthode comprenant :
    la transformation de cellules mammaliennes primaires comprenant ledit gène cible avec une construction comprenant un gène amplifiable et au moins une région le flanquant d'un total d'au moins environ 150 pb homologue avec une séquence d'ADN au lieu de la région de codage dudit gène cible pour produire une amplification dudit gène cible, où ledit gène amplifiable est en un site qui n'interfère pas avec l'expression dudit gène cible, ainsi ladite construction devient intégrée de manière homologue dans le génome desdites cellules primaires pour définir une région amplifiable ;
    la sélection pour des cellules primaires comprenant ladite construction au moyen dudit gène amplifiable ou autre marqueur présent dans ladite construction ;
    l'isolement de portions d'ADN dudit génome desdites cellules primaires, où lesdites portions sont suffisamment grandes pour inclure la totalité de ladite région amplifiable ;
    la transformation des cellules hôtes d'expression secondaire avec lesdites portions d'ADN de cellules primaires et le clonage desdites cellules hôtes d'expression secondaire transformées pour produire des clones desdites cellules hôtes d'expression secondaire différant par lesdites portions d'ADN présentes dans lesdites cellules hôtes d'expression secondaire ;
    la sélection de clones desdites cellules hôtes mammaliennes d'expression secondaire comprenant ladite région amplifiable ; et
    l'amplification de ladite région amplifiable au moyen d'un agent d'amplification, où ladite amplification est avant ledit isolement ou après ladite sélection et avant ladite croissance.
  2. Méthode selon la revendication 1, où au moins une région flanquante a ladite homologie avec une séquence d'ADN dans les limites de 50 kb de la région de codage dudit gène cible.
  3. Méthode selon la revendication 1 ou la revendication 2, où lesdites portions sont des chromosomes en méthaphase.
  4. Méthode selon la revendication 1 ou la revendication 2, où lesdites portions sont des fragments de restriction.
  5. Méthode selon la revendication 1, où ladite construction comprend un marqueur biocide donnant une résistance à un biocide pour lesdites cellules hôtes primaires.
  6. Méthode selon la revendication 1, où ladite construction comprend de plus un gène marqueur séparé de ladite région amplifiable par une région flanquante homologue.
  7. Méthode de production de cellules pour l'expression d'une protéine hétérologue dans une culture, ladite méthode comprenant :
    la transformation de cellules primaires mammaliennes comprenant ledit gène cible avec une construction comprenant un gène amplifiable et au moins une région le flanquant d'au moins environ 150 pb homologue avec une séquence d'ADN à 10 kb de la région codante dudit gène cible, où ledit gène amplifiable est en un site qui n'interfère pas avec l'expression dudit gène cible, ainsi ladite construction se trouve intégrée de façon homologue dans le génome desdites cellules primaires pour définir une région amplifiable comprenant ledit gène amplifiable et ledit gène cible dans ledit génome ;
    la sélection pour des cellules primaires comprenant ladite construction au moyen dudit gène amplifiable ou autre marqueur présent dans ladite construction ;
    l'isolement de portions d'ADN dudit génome desdites cellules primaires où lesdites portions sont suffisamment grandes pour inclure la totalité de ladite région amplifiable ;
    la transformation de cellules hôtes mammaliennes d'expression secondaire avec lesdites portions d'ADN des cellules primaires, où lesdites cellules hôtes d'expression secondaire sont d'une espèce différente desdites cellules hôtes primaires et le clonage desdites cellules hôtes transformées d'expression secondaire pour produire des clones desdites cellules hôtes d'expression secondaire qui diffèrent par lesdites portions d'ADN présentes dans lesdites cellules hôtes d'expression secondaire ;
    la sélection de clones desdites cellules hôtes mammaliennes d'expression secondaire comprenant ladite région amplifiable ; et l'amplification de ladite région amplifiable au moyen d'un agent amplificateur, où ladite amplification est soit avant ledit isolement ou après ladite sélection.
  8. Méthode selon l'une quelconque des revendications précédentes, où ladite amplification est avec lesdites cellules hôtes d'expression secondaire.
  9. Méthode selon l'une quelconque des revendications précédentes, où lesdites cellules primaires sont des cellules humaines.
  10. Méthode selon l'une quelconque des revendications précédentes, où lesdites cellules humaines sont des cellules diploïdes de fibroblaste.
  11. Méthode selon l'une quelconque des revendications précédentes, où le gène amplifiable est un gène mammalien de DHFR.
  12. Méthode selon la revendication 11, où ledit gène amplifiable est un gène muté de DHFR ayant une plus forte valeur de Km que le gène du type sauvage.
  13. Méthode selon la revendication 12, où ladite cellule d'expression d'hôte secondaire est déficiente en DHFR.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7619007B2 (en) 2004-11-23 2009-11-17 Adamas Pharmaceuticals, Inc. Method and composition for administering an NMDA receptor antagonist to a subject

Families Citing this family (253)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6048729A (en) * 1987-05-01 2000-04-11 Transkaryotic Therapies, Inc. In vivo protein production and delivery system for gene therapy
FR2646438B1 (fr) 1989-03-20 2007-11-02 Pasteur Institut Procede de remplacement specifique d'une copie d'un gene present dans le genome receveur par l'integration d'un gene different de celui ou se fait l'integration
EP0747485B1 (fr) * 1989-11-06 1998-12-02 Cell Genesys, Inc. Production de protéines par recombinaison homologue
ES2151463T5 (es) * 1989-12-22 2012-10-29 Merck Serono Sa Constructos de ADN para la activación y la modificación de la expresión de genes endógenos
US5272071A (en) * 1989-12-22 1993-12-21 Applied Research Systems Ars Holding N.V. Method for the modification of the expression characteristics of an endogenous gene of a given cell line
GB9028062D0 (en) * 1990-12-24 1991-02-13 Agricultural & Food Res Production of transgenic animals
DE69233387T2 (de) * 1991-05-06 2005-07-21 Cell Genesys, Inc., Foster City Genmanipulation und Expression mittels genomischer Elemente
EP0587791B1 (fr) * 1991-05-31 2004-04-07 Genentech, Inc. Amelioration de l'expression par ciblage genique dans des sequences endogenes similaires a un retrovirus
AU2515992A (en) * 1991-08-20 1993-03-16 Genpharm International, Inc. Gene targeting in animal cells using isogenic dna constructs
PT101031B (pt) * 1991-11-05 2002-07-31 Transkaryotic Therapies Inc Processo para o fornecimento de proteinas por terapia genetica
US5733761A (en) * 1991-11-05 1998-03-31 Transkaryotic Therapies, Inc. Protein production and protein delivery
US6054288A (en) * 1991-11-05 2000-04-25 Transkaryotic Therapies, Inc. In vivo protein production and delivery system for gene therapy
US5641670A (en) * 1991-11-05 1997-06-24 Transkaryotic Therapies, Inc. Protein production and protein delivery
US6270989B1 (en) 1991-11-05 2001-08-07 Transkaryotic Therapies, Inc. Protein production and delivery
US6692737B1 (en) 1991-11-05 2004-02-17 Transkaryotic Therapies, Inc. In vivo protein production and delivery system for gene therapy
US6063630A (en) 1991-11-05 2000-05-16 Transkaryotic Therapies, Inc. Targeted introduction of DNA into primary or secondary cells and their use for gene therapy
US20020108136A1 (en) * 1997-03-21 2002-08-08 Sri Transgenic animals produced by homologous sequence targeting
WO1993024642A1 (fr) * 1992-06-04 1993-12-09 Exemplar Corporation Insertion d'adn heterologue en dehors de genes chromosomiques connus
US6531124B1 (en) 1992-07-10 2003-03-11 Transkaryotic Therapies, Inc. In vivo production and delivery of insulinotropin for gene therapy
US6670178B1 (en) 1992-07-10 2003-12-30 Transkaryotic Therapies, Inc. In Vivo production and delivery of insulinotropin for gene therapy
US5733753A (en) * 1992-12-22 1998-03-31 Novo Nordisk A/S Amplification of genomic DNA by site specific integration of a selectable marker construct
EP1403376A3 (fr) 1993-04-21 2007-08-29 The University Court Of The University Of Edinburgh Expression de gènes hétérogènes selon un profil d'expression spécifique
US6309853B1 (en) 1994-08-17 2001-10-30 The Rockfeller University Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof
US6471956B1 (en) 1994-08-17 2002-10-29 The Rockefeller University Ob polypeptides, modified forms and compositions thereto
US6350730B1 (en) 1994-08-17 2002-02-26 The Rockefeller University OB polypeptides and modified forms as modulators of body weight
US6048837A (en) * 1994-08-17 2000-04-11 The Rockefeller University OB polypeptides as modulators of body weight
US6001968A (en) 1994-08-17 1999-12-14 The Rockefeller University OB polypeptides, modified forms and compositions
US6124448A (en) * 1994-08-17 2000-09-26 The Rockfeller University Nucleic acid primers and probes for the mammalian OB gene
US6124439A (en) * 1994-08-17 2000-09-26 The Rockefeller University OB polypeptide antibodies and method of making
US5759775A (en) 1994-10-27 1998-06-02 Genetech, Inc. Methods for detecting nucleic acids encoding AL--1 neurotrophic factor
US6610296B2 (en) 1995-10-26 2003-08-26 Genentech, Inc. Methods of enhancing cognitive function using an AL-1 neurotrophic factor immunoadhesin
IL125073A0 (en) 1996-01-08 1999-01-26 Genentech Inc Wsx receptor and ligands
US7063958B1 (en) 1996-01-16 2006-06-20 The Rockefeller University Nucleic acids db, the receptor for leptin
US7148004B1 (en) 1997-01-16 2006-12-12 The Rockefeller University Oligonucleotides of the OB-R isoforms and methods of diagnosing body weight
US7084252B1 (en) 1996-01-16 2006-08-01 The Rockefeller University DB, the receptor for leptin
US7619079B2 (en) 1996-02-14 2009-11-17 The Rockefeller University Db, the receptor for leptin, nucleic acids encoding the receptor, and uses thereof
US5792210A (en) * 1996-06-10 1998-08-11 Environmental Behavior Modification Inc. Electrical tongue stimulator and method for addiction treatment
JP2000517185A (ja) 1996-08-29 2000-12-26 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 新規なメタロプロテアーゼファミリーｋｕｚ
US6566089B1 (en) * 1996-09-04 2003-05-20 Tularik Inc. Cell-based drug screens for regulators of gene expression
EP0966521A1 (fr) * 1996-09-13 1999-12-29 Novo Nordisk Biotech, Inc. Cellules presentant des mutations d'insertion d'adn produisant des quantites modifiees d'un polypeptide
JPH10117771A (ja) * 1996-10-23 1998-05-12 Natl Food Res Inst 冷凍生地耐性実用パン酵母
US5925544A (en) * 1996-11-18 1999-07-20 Novo Nordisk A/S Method of homologous recombination followed by in vivo selection of DNA amplification
DK1717322T3 (da) 1997-01-17 2012-10-22 Codexis Mayflower Holdings Llc Udvikling af hele celler og organismer ved rekursiv sekvensrekombination
US7148054B2 (en) * 1997-01-17 2006-12-12 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US6326204B1 (en) 1997-01-17 2001-12-04 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
US6372453B1 (en) 1997-02-18 2002-04-16 Genetech, Inc. Neurturin receptor
US6025157A (en) * 1997-02-18 2000-02-15 Genentech, Inc. Neurturin receptor
US6777196B2 (en) 1997-02-18 2004-08-17 Genentech, Inc. Neurturin receptor
PL191251B1 (pl) 1997-03-14 2006-04-28 Idec Pharma Corp Sposób wprowadzania żądanego DNA w docelowe miejsce w genomie ssaka i układ wektorów
US5948653A (en) 1997-03-21 1999-09-07 Pati; Sushma Sequence alterations using homologous recombination
US6172211B1 (en) 1997-07-11 2001-01-09 Boehringer Ingelheim International Gmbh Nucleic acid encoding tag7 polypeptide
DK0986644T3 (da) * 1997-07-23 2007-01-29 Roche Diagnostics Gmbh Fremstilling af erythropoietin ved endogen genaktivering med virale promotorer
PT986644E (pt) 1997-07-23 2007-01-31 Roche Diagnostics Gmbh Preparação de eritropoietina por activação genética endógena com promotores virais
US6548296B1 (en) 1997-07-23 2003-04-15 Roche Diagnostics Gmbh Methods for identifying human cell lines useful for endogenous gene activation, isolated human lines identified thereby, and uses thereof
US6395484B1 (en) * 1997-07-23 2002-05-28 Roche Diagnostics Gmbh Identification of human cell lines for the production of human proteins by endogenous gene activation
PT1000154E (pt) 1997-07-23 2007-03-30 Roche Diagnostics Gmbh Identificação de linhas celulares humanas para a produção de proteínas humanas por activação endógena de genes
US6897066B1 (en) 1997-09-26 2005-05-24 Athersys, Inc. Compositions and methods for non-targeted activation of endogenous genes
DE69841723D1 (de) * 1997-09-26 2010-07-29 Abt Holding Co Expression von endogenen genen durch homologe rekombination eines vektorkonstruktes mit zellulärer dna
US7316923B1 (en) 1997-09-26 2008-01-08 Athersys, Inc. Compositions and methods for non-targeted activation of endogenous genes
ZA989497B (en) † 1997-10-20 2000-04-19 Roche Diagnostics Gmbh Positive-negative selection in homologous recombination.
KR100769103B1 (ko) 1997-11-28 2007-10-23 세로노 제네틱스 인스티튜트 에스.에이. 클라미디아 트라코마티스 게놈 서열과 폴리펩티드, 이의단편 및 이의 용도, 특히, 감염의 진단, 예방 및 치료 용도
AU757930B2 (en) * 1997-12-01 2003-03-13 Roche Diagnostics Gmbh Optimization of cells for endogenous gene activation
JPH11243944A (ja) * 1998-03-04 1999-09-14 Natl Food Res Inst 製パン用乾燥耐性実用パン酵母
US6426191B1 (en) 1998-04-03 2002-07-30 Hyseq, Inc. Assays involving an IL-1 receptor antagonist
US6541623B1 (en) 1998-04-03 2003-04-01 Hyseq, Inc. Interleukin—1 receptor antagonist and uses thereof
US6294655B1 (en) 1998-04-03 2001-09-25 Hyseq, Inc. Anti-interleukin-1 receptor antagonist antibodies and uses thereof
US6337072B1 (en) 1998-04-03 2002-01-08 Hyseq, Inc. Interleukin-1 receptor antagonist and recombinant production thereof
KR100795626B1 (ko) 1998-07-21 2008-01-17 코브라 바이오매뉴팩쳐링 피엘씨. 도처에 있는 염색질 개방 요소(유씨오이)를 포함하는폴리뉴클레오티드
WO2000023594A1 (fr) * 1998-10-22 2000-04-27 The General Hospital Corporation PEPTIDES ET DERIVES PEPTIDIQUES BIOACTIFS DE L'HORMONE PARATHYROIDE (PTH) ET DE LA PROTEINE LIEE A L'HORMONE PARATHYROIDE (PTHrP)
US6773911B1 (en) 1998-11-23 2004-08-10 Amgen Canada Inc. Apoptosis-inducing factor
TW570977B (en) * 1998-12-07 2004-01-11 Li-Wei Hsu An expression system for producing recombinant human erythropoietin, method for purifying secreted human erythropoietin and uses thereof
DE19857609A1 (de) 1998-12-14 2000-06-15 Hannelore Ehrenreich Verwendung von Erythropoietin zur Behandlung von cerebralen Ischämien des Menschen
PT2067788E (pt) 1999-05-18 2015-10-29 Dyax Corp Bibliotecas de fragmentos fab e métodos para a sua utilização
US6495360B1 (en) * 1999-05-28 2002-12-17 Photogen, Inc. Method for enhanced protein stabilization and for production of cell lines useful for production of such stabilized proteins
US20050129669A1 (en) * 1999-06-21 2005-06-16 Transkaryotic Therapies, Inc., A Massachusetts Corporation DNA construct for effecting homologous recombination and uses thereof
US7291714B1 (en) 1999-06-30 2007-11-06 Millennium Pharmaceuticals, Inc. Glycoprotein VI and uses thereof
AU7734800A (en) * 1999-09-29 2001-04-30 General Hospital Corporation, The Polypeptide derivatives of parathyroid hormone (pth)
EP1222208B1 (fr) 1999-09-29 2008-10-29 The General Hospital Corporation Derives polypeptides de parathormone (pth)
AU1647501A (en) 1999-12-10 2001-06-18 Cytos Biotechnology Ag Replicon based activation of endogenous genes
EP1266016A2 (fr) * 2000-01-13 2002-12-18 Novozymes Biotech, Inc. Procedes de production d'un polypeptide au moyen d'une sequence d'initiateurs de traduction degrades
EP1254230A2 (fr) 2000-02-29 2002-11-06 Millennium Pharmaceuticals, Inc. Recepteurs couples a une proteine g, numerotees 1983, 52881, 2398, 45449, 50289, et 52872, et utilisations correspondantes
EP1714661A3 (fr) 2000-05-19 2012-03-14 The Center for Blood Research, INC. Procedes pour diagnostiquer et traiter des troubles hemostatiques par modulation de l'activité de la p-selectine
US6656700B2 (en) 2000-05-26 2003-12-02 Amersham Plc Isoforms of human pregnancy-associated protein-E
US6686188B2 (en) 2000-05-26 2004-02-03 Amersham Plc Polynucleotide encoding a human myosin-like polypeptide expressed predominantly in heart and muscle
GB0018876D0 (en) 2000-08-01 2000-09-20 Applied Research Systems Method of producing polypeptides
AU2001284703B2 (en) * 2000-08-03 2007-03-22 Therapeutic Human Polyclonals Inc. Production of humanized antibodies in transgenic animals
US6803211B2 (en) 2000-08-25 2004-10-12 Pfizer Inc. Methods and compositions for diagnosing and treating disorders involving angiogenesis
US7311905B2 (en) 2002-02-13 2007-12-25 Anthrogenesis Corporation Embryonic-like stem cells derived from post-partum mammalian placenta, and uses and methods of treatment using said cells
EP2322601A1 (fr) 2000-12-06 2011-05-18 Anthrogenesis Corporation Procédé de collecte de cellules souche placentaires
WO2002049673A2 (fr) 2000-12-20 2002-06-27 F. Hoffmann-La Roche Ag Conjugues d'erythropoietine
AU3323002A (en) * 2000-12-20 2002-07-01 Hoffmann La Roche Erythropoietin conjugates
PA8536201A1 (es) 2000-12-29 2002-08-29 Kenneth S Warren Inst Inc Protección y mejoramiento de células, tejidos y órganos que responden a la eritropoyetina
EP2338983B1 (fr) 2001-02-14 2015-10-07 Anthrogenesis Corporation Renovation et repeuplement de tissus decellularises er d'organes preleves sur des cadavres par des cellules souches.
CA2438153C (fr) 2001-02-14 2015-06-02 Anthrogenesis Corporation Placenta post-gravidique de mammifere, son utilisation et cellules souches placentaires correspondantes
US8231878B2 (en) 2001-03-20 2012-07-31 Cosmo Research & Development S.P.A. Receptor trem (triggering receptor expressed on myeloid cells) and uses thereof
US8981061B2 (en) 2001-03-20 2015-03-17 Novo Nordisk A/S Receptor TREM (triggering receptor expressed on myeloid cells) and uses thereof
EP1925671A1 (fr) 2001-06-06 2008-05-28 Bioriginal Food & Science Corp. Promoteurs spécifiques à la graine lin (Linum usitatissimum L.)
ATE404672T1 (de) 2001-06-06 2008-08-15 Bioriginal Food & Science Corp Samen-spezifische sequenzen aus flachs (linum usitatissimum l.)
AU2002339843B2 (en) * 2001-07-23 2007-12-06 The General Hospital Corporation Conformationally constrained parathyroid hormone (PTH) analogs
KR20040066788A (ko) 2001-08-31 2004-07-27 더 락커펠러 유니버시티 프스포다이에스터레이즈 활성 및 뇌에서프스포다이에스터레이즈 1ｂ 매개되는 신호의 조절
US20040142325A1 (en) 2001-09-14 2004-07-22 Liat Mintz Methods and systems for annotating biomolecular sequences
US20030170691A1 (en) 2001-12-19 2003-09-11 Millennium Pharmaceuticals, Inc. Human diacylglycerol acyltransferase 2 (DGAT2) family members and uses therefor
KR100982922B1 (ko) * 2002-01-17 2010-09-20 론자 바이올로직스 피엘씨 단백질을 생산할 수 있으며 무글루타민 배지에서 성장할 수있는 글루타민-요구성 인간세포
US7682803B2 (en) 2005-10-13 2010-03-23 Anthrogenesis Corporation Immunomodulation using placental stem cells
EP2301343A1 (fr) 2002-02-13 2011-03-30 Anthrogenesis Corporation Cellules souches de type embryonnaire dérivées de placenta mammalien post-partum, utilisations, et procédés de traitement à base de cellules de ce type
US7498171B2 (en) 2002-04-12 2009-03-03 Anthrogenesis Corporation Modulation of stem and progenitor cell differentiation, assays, and uses thereof
AU2003241359A1 (en) * 2002-05-06 2003-11-11 Functional Genetics, Inc. Mammalian genes involed in rapamycin resistance and tumorgenesis: annexin xiii genes
AU2003239589A1 (en) 2002-05-24 2003-12-12 Schering-Plough Ltd. Eta-1 gene and methods for use
US20060099676A1 (en) * 2002-08-15 2006-05-11 Limin Li Mammalian genes involved in rapamycin resistance and tumorgenesis: rapr6 genes
WO2004020581A2 (fr) * 2002-08-15 2004-03-11 Functional Genetics, Inc. Genes mammaliens impliques dans la resistance a la rapamycine et la genese tumorale: genes rapr7
US7459435B2 (en) 2002-08-29 2008-12-02 Hoffmann-La Roche Inc. Treatment of disturbances of iron distribution
AU2003293013A1 (en) 2002-11-21 2004-06-18 University Of Massachusets Diagnosing and treating hematopoietic cancers
US7459436B2 (en) 2002-11-22 2008-12-02 Hoffmann-La Roche Inc. Treatment of disturbances of iron distribution
EP2112229A3 (fr) 2002-11-25 2009-12-02 Sequenom, Inc. Procédés d'identification du risque du cancer du sein et traitements associés
KR20050086780A (ko) 2002-11-26 2005-08-30 안트로제네시스 코포레이션 세포요법제, 세포요법제 단위 및 이를 이용한 치료방법
EP1587545A2 (fr) * 2003-01-13 2005-10-26 Mahendra S. Rao Molecule candidate a expression persistante dans des cellules souches et progenitrices destinee a l'apport de produits therapeutiques
US20050048041A1 (en) * 2003-01-13 2005-03-03 Rao Mahendra S. Persistent expression of candidate molecule in proliferating stem and progenitor cells for delivery of therapeutic products
EP2561887B8 (fr) 2003-01-14 2016-12-21 Dana-Farber Cancer Institute, Inc. Sensibilisateur de thérapie anticancéreuse
US8021833B2 (en) 2003-02-12 2011-09-20 Functional Genetics, Inc. Method for reducing HIV viral budding by administering a VPS28-specfic antibody that disrupts Gag-TSG101-VPS28 binding interactions
US7795220B2 (en) * 2003-03-19 2010-09-14 The General Hospital Corporation Conformationally constrained parathyroid hormones with alpha-helix stabilizers
EP1653985A4 (fr) 2003-07-17 2009-08-05 Gen Hospital Corp Analogues d'hormone parathyroide (pth) a conformation contrainte
US7287952B2 (en) * 2003-07-23 2007-10-30 Lockheed Martin Corporation Feeder load automation system and method of use
US8409861B2 (en) 2003-08-08 2013-04-02 Sangamo Biosciences, Inc. Targeted deletion of cellular DNA sequences
US11311574B2 (en) 2003-08-08 2022-04-26 Sangamo Therapeutics, Inc. Methods and compositions for targeted cleavage and recombination
US7888121B2 (en) 2003-08-08 2011-02-15 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
EP1670492A4 (fr) 2003-09-29 2009-07-08 Warren Pharmaceuticals Inc Cytokines de protection des tissus pour le traitement et la prevention des septicemies et de la formation d'adhesions
BRPI0416141B8 (pt) 2003-11-01 2021-05-25 Biovation Ltd anticorpo anti-cd52 modificado, composição farmacêutica, vetores de expressão, e método para preparar uma imunoglobulina
PL1696947T3 (pl) * 2003-12-19 2014-08-29 Hoffmann La Roche Zastosowanie erytropoetyny w leczeniu zaburzeń dystrybucji żelaza w przewlekłych chorobach zapalnych jelit
ES2315842T3 (es) * 2004-01-19 2009-04-01 Nsgene A/S Celulas terapeuticas humanas que secretan factor de crecimiento nervioso.
WO2006017621A2 (fr) 2004-08-02 2006-02-16 Janssen Pharmaceutica N.V. Variant d'epissage d'irak1c et son utilisation
US7893197B2 (en) 2004-08-25 2011-02-22 Janssen Pharmaceutica N.V. Relaxin-3 chimeric polypeptides and their preparation and use
CA2583375C (fr) 2004-09-02 2013-01-15 Yale University Regulation d'oncogenes par des micro-arn
US20060130157A1 (en) 2004-10-22 2006-06-15 Kevin Wells Ungulates with genetically modified immune systems
GB0426146D0 (en) 2004-11-29 2004-12-29 Bioxell Spa Therapeutic peptides and method
EP1844068A4 (fr) * 2005-01-25 2009-09-30 Apollo Life Sciences Ltd Molécules et leurs molécules chimériques
US8088978B2 (en) 2005-02-09 2012-01-03 Bioriginal Food & Science Corp. Omega-3 fatty acid desaturase family members and uses thereof
MX2007012374A (es) 2005-04-06 2008-02-22 Adamas Pharmaceuticals Inc Metodos y composiciones para el tratamiento de condiciones relacionadas con cns.
US8354384B2 (en) 2005-06-23 2013-01-15 Yale University Anti-aging micrornas
US7767420B2 (en) 2005-11-03 2010-08-03 Momenta Pharmaceuticals, Inc. Heparan sulfate glycosaminoglycan lyase and uses thereof
US8014957B2 (en) 2005-12-15 2011-09-06 Fred Hutchinson Cancer Research Center Genes associated with progression and response in chronic myeloid leukemia and uses thereof
CN101389754A (zh) 2005-12-29 2009-03-18 人类起源公司 胎盘干细胞和第二来源干细胞的联合培养
CN101395266B (zh) 2005-12-29 2018-06-15 人类起源公司 胎盘干细胞群
DE102006004008A1 (de) 2006-01-27 2007-08-02 Hannelore Prof. Dr. Dr. Ehrenreich Verfahren zur Behandlung und/oder Prophylaxe von Multipler Sklerose, sowie Verwendung von Erythropoietin zur Herstellung eines Arzneimittels zur intermittierenden Behandlung und/oder intermittierenden Prophylaxe von Multipler Sklerose
TWI428448B (zh) 2006-03-24 2014-03-01 Syntonix Pharmaceuticals Inc 作為第九因子（ｆａｃｔｏｒ ｉｘ）原肽處理酶之ｐｃ５
US10522240B2 (en) 2006-05-03 2019-12-31 Population Bio, Inc. Evaluating genetic disorders
US7702468B2 (en) 2006-05-03 2010-04-20 Population Diagnostics, Inc. Evaluating genetic disorders
WO2007140599A1 (fr) 2006-06-05 2007-12-13 Cancer Care Ontario Évaluation de risque pour cancer colorectal
WO2008019214A1 (fr) * 2006-08-04 2008-02-14 Prolong Pharmaceuticals, Inc. Erythropoïétine modifiée
JP2009545320A (ja) * 2006-08-04 2009-12-24 ザ ジェネラル ホスピタル コーポレイション 副甲状腺ホルモン（ｐｔｈ）のポリペプチド誘導体
JP5087625B2 (ja) 2006-09-01 2012-12-05 セラピューティック・ヒューマン・ポリクローナルズ・インコーポレーテッド 非ヒトトランスジェニック動物におけるヒトまたはヒト化免疫グロブリンの発現強化
CN101631852A (zh) 2006-10-23 2010-01-20 人类起源公司 用胎盘细胞群治疗骨缺损的方法和组合物
WO2008133711A2 (fr) 2006-10-30 2008-11-06 Functional Genetics, Inc. Perturbation génétique homozygote aléatoire pour améliorer la production d'anticorps
EP2630959A1 (fr) 2007-02-12 2013-08-28 Anthrogenesis Corporation Traitement de maladies inflammatoires au moyen de cellules souches placentaires
US20110189663A1 (en) 2007-03-05 2011-08-04 Cancer Care Ontario Assessment of risk for colorectal cancer
EP2977452A3 (fr) 2007-05-11 2016-05-25 Thomas Jefferson University Procédés de traitement et de prévention de maladies et de troubles neurodégénératifs
MX2009012197A (es) 2007-05-11 2010-01-15 Univ Pennsylvania Metodos para tratamiento de ulceras en la piel.
AU2008279550B2 (en) 2007-06-21 2012-08-09 Angelica Therapeutics, Inc. Modified toxins
CL2008002054A1 (es) 2007-07-17 2009-05-29 Hoffmann La Roche Metodo para la regeneracion de una columna de cromatografia de intercambio cationico despues de la elusion de eritropoyetina monopeguilada y metodo para obtener una eritropoyetina monopeguilada, incorporando el metodo de regeneracion de la columna de intercambio cationico.
AR067537A1 (es) 2007-07-17 2009-10-14 Hoffmann La Roche Purificacion de polipeptidos pegilados
WO2009010107A1 (fr) 2007-07-19 2009-01-22 Hannelore Ehrenreich Utilisation d'activation ou de stimulation du récepteur de l'epo pour l'amélioration du score edss dans des patients présentant une sclérose en plaques
AU2008282805B2 (en) 2007-08-01 2014-05-01 Chugai Pharmaceutical Co., Ltd. Screening methods using G-protein coupled receptors and related compositions
US9200253B1 (en) 2007-08-06 2015-12-01 Anthrogenesis Corporation Method of producing erythrocytes
WO2009042538A2 (fr) 2007-09-24 2009-04-02 Cornell University Protéines immunogènes provenant d'une membrane extérieure dérivée du génome de leptospira et compositions et procédés à base de celles-ci
KR20210127819A (ko) 2007-09-28 2021-10-22 안트로제네시스 코포레이션 인간 태반 관류액 및 인간 태반-유래 중간체 천연 킬러 세포를 사용한 종양 억제 방법
US8501449B2 (en) 2007-12-04 2013-08-06 Proteon Therapeutics, Inc. Recombinant elastase proteins and methods of manufacturing and use thereof
EP2570499A3 (fr) 2008-01-02 2013-04-03 Suregene Llc Marqueurs génétiques de maladie mentale
CA2712075A1 (fr) 2008-01-17 2009-07-23 Suregene Llc Marqueurs genetiques de maladie mentale
WO2009111375A2 (fr) * 2008-03-01 2009-09-11 Abraxis Bioscience, Llc Traitement, diagnostic et procédé pour découvrir un antagoniste à l'aide d'arnmi spécifique de sparc
JP5670755B2 (ja) 2008-03-12 2015-02-18 ザ ロックフェラー ユニバーシティ 翻訳プロファイリング及び分子表現型解析のための方法及び組成物
WO2009137660A2 (fr) * 2008-05-07 2009-11-12 Abraxis Bioscience, Llc Amélioration de pharmacothérapie par précurseurs arn
KR20210010648A (ko) 2008-08-20 2021-01-27 안트로제네시스 코포레이션 개선된 세포 조성물 및 그의 제조 방법
US8476013B2 (en) 2008-09-16 2013-07-02 Sequenom, Inc. Processes and compositions for methylation-based acid enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
US8962247B2 (en) 2008-09-16 2015-02-24 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non invasive prenatal diagnoses
EP2344672B8 (fr) 2008-09-25 2014-12-24 SureGene LLC Marqueurs génétiques pour optimiser le traitement de la schizophrénie
CA2998281C (fr) 2008-09-26 2022-08-16 Dana-Farber Cancer Institute, Inc. Anticorps anti-pd-1 humains et leurs utilisations
WO2010037224A1 (fr) * 2008-10-03 2010-04-08 St. Michael's Hospital Procédé de prévention et de traitement de maladies cardiovasculaires avec brca1
US20100120063A1 (en) * 2008-10-10 2010-05-13 Discoverx Corporation GPCR Arrestin Assays
ES2548377T3 (es) 2008-10-27 2015-10-16 Revivicor, Inc. Ungulados inmunodeprimidos
EA201170647A1 (ru) 2008-11-04 2011-12-30 Янссен Фармацевтика Нв Пептиды-агонисты crhr2 и их использование
EP2367932B1 (fr) 2008-11-19 2019-06-12 Celularity, Inc. Cellules adhérentes issues de l'amnios
JP2012515532A (ja) 2009-01-20 2012-07-12 ラモット アット テル アビブ ユニバーシティ， リミテッド Ｍｉｒ−２１プロモーター駆動性標的がん治療
US8530629B2 (en) 2009-01-30 2013-09-10 Ab Biosciences, Inc. Lowered affinity antibodies and uses therefor
US8211655B2 (en) * 2009-02-12 2012-07-03 Discoverx Corporation Wild-type receptor assays
MX2012005262A (es) 2009-11-04 2012-09-28 Janssen Pharmaceutica Nv Metodo para tratar la insuficiencia cardiaca con petidos tipo estrescopina.
EP2499159B1 (fr) 2009-11-13 2017-01-04 Dana-Farber Cancer Institute, Inc. Compositions, kits et méthodes utilisés pour le diagnostic, le pronostic, la surveillance, le traitement et la modulation des lymphoproliférations après greffe d'organe et de l'hypoxie associée à l'angiogenèse au moyen de la galectine-1
CN102770529B (zh) 2009-11-17 2018-06-05 Musc研究发展基金会 针对人核仁素的人单克隆抗体
WO2011087760A2 (fr) 2009-12-22 2011-07-21 Sequenom, Inc. Procédés et kits pour identifier une aneuploïdie
WO2011094181A1 (fr) 2010-01-26 2011-08-04 Anthrogenesis Corporation Traitement de cancers associés aux os utilisant des cellules souches placentaires
US9624290B2 (en) 2010-01-28 2017-04-18 Ab Biosciences, Inc. Lowered affinity antibodies and methods of making the same
EP2552947A4 (fr) 2010-03-26 2013-11-13 Dartmouth College Vista, une protéine médiatrice régulatrice des lymphocytes t, agents de liaison à vista et utilisation associée
US10745467B2 (en) 2010-03-26 2020-08-18 The Trustees Of Dartmouth College VISTA-Ig for treatment of autoimmune, allergic and inflammatory disorders
US20150231215A1 (en) 2012-06-22 2015-08-20 Randolph J. Noelle VISTA Antagonist and Methods of Use
CN107699541A (zh) 2010-04-07 2018-02-16 人类起源公司 使用胎盘干细胞的血管生成
RU2604809C2 (ru) 2010-05-13 2016-12-10 Дзе Дженерал Хоспитал Корпорейшн Аналоги паратиреоидного гормона и их применение
KR20200077613A (ko) 2010-07-13 2020-06-30 안트로제네시스 코포레이션 천연 킬러 세포의 생성 방법
DK2601609T3 (en) 2010-08-02 2017-06-06 Population Bio Inc COMPOSITIONS AND METHODS FOR DISCOVERING MUTATIONS CAUSING GENETIC DISORDERS
CA2808748C (fr) 2010-09-14 2018-09-11 Roberto Falkenstein Procede pour purifier l'erythropoietine pegylee
US20120310140A1 (en) 2010-12-01 2012-12-06 Spinal Modulation, Inc. Directed delivery of agents to neural anatomy
EP2471543A1 (fr) 2010-12-02 2012-07-04 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Induction de tolérance ou immunosuppression pour empêcher en particulier la réaction du greffon contre l'hôte (GvHD) par une pré-incubation de courte durée des suspensions de cellules transplantées, tissus ou organes revêtus par les ligands sur les molécules à la surface des cellules
WO2012092485A1 (fr) 2010-12-31 2012-07-05 Anthrogenesis Corporation Amélioration de l'efficacité de cellules souches placentaires sous l'effet de molécules d'arn modulateur
ES2707579T3 (es) 2011-06-01 2019-04-04 Celularity Inc Tratamiento del dolor usando citoblastos placentarios
US8759313B2 (en) 2011-08-03 2014-06-24 The Charlotte-Mecklenburg Hospital Authority Treatment of fibrosis using microRNA 19b
US20140227293A1 (en) 2011-06-30 2014-08-14 Trustees Of Boston University Method for controlling tumor growth, angiogenesis and metastasis using immunoglobulin containing and proline rich receptor-1 (igpr-1)
CA2851388C (fr) 2011-10-10 2023-11-21 The Hospital For Sick Children Methodes et compositions de depistage et de traitement de troubles du developpement
DK2773779T3 (da) 2011-11-04 2020-11-23 Population Bio Inc Fremgangsmåder og sammensætninger til diagnosticering, prognose og forebyggelse af neurologiske tilstande
DK2812452T3 (da) 2012-02-09 2020-06-29 Population Bio Inc Fremgangsmåder og sammensætninger til screening og behandling af udviklingsforstyrrelser
US9550830B2 (en) 2012-02-15 2017-01-24 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
EP2814842B1 (fr) 2012-02-15 2018-08-22 Novo Nordisk A/S Anticorps liant peptidoglycane reconnaissance protéine 1
EP2814844B1 (fr) 2012-02-15 2017-08-02 Novo Nordisk A/S Anticorps qui se lient et bloquent la stimulation d'un récepteur exprimé sur des cellules myéloïdes 1 (trem-1)
ES2930180T3 (es) 2012-03-02 2022-12-07 Sequenom Inc Métodos para enriquecer ácido nucleico canceroso a partir de una muestra biológica
WO2013152011A1 (fr) 2012-04-02 2013-10-10 Ab Biosciences, Inc. Nouveaux anticorps témoins humains et leurs utilisations
DK2839013T3 (da) * 2012-04-18 2020-09-14 Univ Leland Stanford Junior Ikke-disruptiv-gen-targetering
US9920361B2 (en) 2012-05-21 2018-03-20 Sequenom, Inc. Methods and compositions for analyzing nucleic acid
US9890215B2 (en) 2012-06-22 2018-02-13 King's College London Vista modulators for diagnosis and treatment of cancer
CA2878979C (fr) 2012-07-13 2021-09-14 Sequenom, Inc. Procedes et compositions pour enrichissement base sur la methylation en acide nucleique foetal dans un echantillon maternel, utiles pour les diagnostics prenatals non invasifs
CA2884704C (fr) 2012-09-07 2023-04-04 Randolph J. Noelle Modulateurs vista de diagnostic et de traitement de cancer
WO2014043519A1 (fr) 2012-09-14 2014-03-20 Population Diagnostics Inc. Procédés et compositions pour le diagnostic, le pronostic et le traitement d'états neurologiques
EP2900835A4 (fr) 2012-09-27 2016-05-11 Population Diagnotics Inc Procédés et compositions de dépistage et de traitement de troubles du développement
CN103864913A (zh) 2012-12-18 2014-06-18 联亚生技开发股份有限公司 重组蛋白
JP2016506968A (ja) 2013-02-05 2016-03-07 アントフロゲネシス コーポレーション 胎盤由来のナチュラルキラー細胞
WO2014168711A1 (fr) 2013-03-13 2014-10-16 Sequenom, Inc. Amorces pour analyse de la méthylation de l'adn
EP2970420A4 (fr) 2013-03-15 2016-08-17 Apotex Inc Stabilité de la formulation liquide améliorée d'érythropoïétine alpha par l'intermédiaire d'un traitement de purification
JP2016519651A (ja) 2013-03-15 2016-07-07 アンジェリカ セラピューティックス，インク． 改質された毒素
US10294295B2 (en) 2013-11-27 2019-05-21 Inis Biotech Llc Methods for modulating angiogenesis of cancers refractory to anti-VEGF treatment
CN106661107B (zh) 2013-12-24 2021-12-24 杨森制药公司 抗vista抗体及片段
US11014987B2 (en) 2013-12-24 2021-05-25 Janssen Pharmaceutics Nv Anti-vista antibodies and fragments, uses thereof, and methods of identifying same
EP3736344A1 (fr) 2014-03-13 2020-11-11 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
AU2015231231B2 (en) 2014-03-21 2021-09-02 The Board Of Trustees Of The Leland Stanford Junior University Genome editing without nucleases
WO2015191881A2 (fr) 2014-06-11 2015-12-17 Green Kathy A Utilisation d'antagonistes et d'agonistes vista pour supprimer ou améliorer l'immunité humorale
US10724096B2 (en) 2014-09-05 2020-07-28 Population Bio, Inc. Methods and compositions for inhibiting and treating neurological conditions
KR102473092B1 (ko) 2014-09-15 2022-12-01 칠드런'즈 메디컬 센터 코포레이션 히스톤 h3-리신 트리메틸화를 제거함으로써 체세포 핵 이식(scnt) 효율을 증가시키는 방법 및 조성물
EP3015475A1 (fr) 2014-10-31 2016-05-04 Novartis AG Cellules de mammifère exprimant des antigènes du cytomégalovirus
CN107405398A (zh) 2014-12-05 2017-11-28 伊穆奈克斯特股份有限公司 鉴定vsig8作为推定vista受体及其用以产生vista/vsig8激动剂和拮抗剂的用途
EP3031822A1 (fr) 2014-12-08 2016-06-15 Novartis AG Antigènes du cytomégalovirus
EP3048114A1 (fr) 2015-01-22 2016-07-27 Novartis AG Antigènes du cytomégalovirus et leurs utilisations
US10632151B2 (en) 2015-01-22 2020-04-28 University Of Massachusetts Cancer immunotherapy
EP3047856A1 (fr) 2015-01-23 2016-07-27 Novartis AG Antigènes de cytomégalovirus (cmv) et leurs utilisations
DK3313882T3 (da) 2015-06-24 2020-05-11 Janssen Pharmaceutica Nv Anti-VISTA antistoffer og fragmenter
CA3014013A1 (fr) 2016-02-12 2017-08-17 Janssen Pharmaceutica Nv Anticorps anti-vista (b7h5)
WO2017156032A1 (fr) 2016-03-07 2017-09-14 Charlestonpharma, Llc Anticorps anti-nucléoline
CR20180537A (es) 2016-04-15 2019-03-04 Immunext Inc Anticuerpos vista antihumanos y su uso
PL3484911T3 (pl) 2016-07-15 2021-03-08 F. Hoffmann-La Roche Ag Sposób oczyszczania PEGylowanej erytropoetyny
WO2018144807A2 (fr) * 2017-02-02 2018-08-09 Massachusetts Institute Of Technology Levure modifiée en tant que procédé de bioremédiation
US10240205B2 (en) 2017-02-03 2019-03-26 Population Bio, Inc. Methods for assessing risk of developing a viral disease using a genetic test
EP3731872B1 (fr) 2017-12-29 2021-11-24 F. Hoffmann-La Roche AG Procédé de fournir des protéines monopégylée
EP3731873B1 (fr) 2017-12-29 2022-01-26 F. Hoffmann-La Roche AG Procédé de fournir des protéines monopégylée
EP3731871B1 (fr) 2017-12-29 2023-10-04 F. Hoffmann-La Roche AG Procédé de fournir des protéines pégylée
AU2019316556C1 (en) 2018-08-08 2024-09-12 Pml Screening, Llc Methods for assessing the risk of developing progressive multifocal leukoencephalopathy caused by john cunningham virus by genetic testing
EP4004018A1 (fr) 2019-07-24 2022-06-01 GlaxoSmithKline Biologicals SA Protéines de cytomégalovirus humain modifiées
WO2022159414A1 (fr) 2021-01-22 2022-07-28 University Of Rochester Érythropoïétine pour un dysfonctionnement gastrointestinal
WO2023144665A1 (fr) 2022-01-28 2023-08-03 Glaxosmithkline Biologicals Sa Protéines de cytomégalovirus humain modifiées
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Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CH634437A5 (fr) 1979-03-14 1983-01-31 Tokyo Shibaura Electric Co Resistance de decharge.
US4634665A (en) * 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4656134A (en) * 1982-01-11 1987-04-07 Board Of Trustees Of Leland Stanford Jr. University Gene amplification in eukaryotic cells
AU570940B2 (en) * 1982-11-30 1988-03-31 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Process for producing poxvirus recombinants for expression offoreign genes
EP0320500B1 (fr) * 1983-01-13 2004-11-17 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Système de vecteur plasmidique Ti non oncogène et molécules d'ADN recombinant pour l'introduction de gènes exprimables dans les gènomes de cellules végétales
US4740461A (en) * 1983-12-27 1988-04-26 Genetics Institute, Inc. Vectors and methods for transformation of eucaryotic cells
EP0273085A1 (fr) * 1986-12-29 1988-07-06 IntraCel Corporation Procédé pour faire entrer des acides nucléiques dans des cellules eucaryotes
US4950599A (en) * 1987-01-29 1990-08-21 Wolf Bertling Method for exchanging homologous DNA sequences in a cell using polyoma encapsulated DNA fragments
ZA88319B (en) * 1987-02-06 1988-08-12 Lubrizol Enterprises, Inc. Ocs enhancer
EP0317509A3 (fr) * 1987-11-16 1990-01-31 Ciba-Geigy Ag Incorporation ciblée de gènes dans le génôme végétal
GB8807683D0 (en) * 1988-03-31 1988-05-05 Ici Plc Regulation of gene expression
ATE129289T1 (de) * 1988-12-08 1995-11-15 Abbott Lab Verfahren zur induzierbaren genexpression unter verwendung eines dhfr-gens.
FR2646438B1 (fr) * 1989-03-20 2007-11-02 Pasteur Institut Procede de remplacement specifique d'une copie d'un gene present dans le genome receveur par l'integration d'un gene different de celui ou se fait l'integration
WO1990014092A1 (fr) * 1989-05-18 1990-11-29 Cell Genesys, Inc. Utilisation d'oligonucleotides mono-brins pour la modification axee sur le site de genes mammiferes in vivo
NL8901931A (nl) * 1989-07-26 1991-02-18 Mogen International N V En Rij Werkwijze voor het plaatsgericht introduceren van vreemd dna in het genoom van planten.
EP0747485B1 (fr) * 1989-11-06 1998-12-02 Cell Genesys, Inc. Production de protéines par recombinaison homologue
US5272071A (en) * 1989-12-22 1993-12-21 Applied Research Systems Ars Holding N.V. Method for the modification of the expression characteristics of an endogenous gene of a given cell line
ES2151463T5 (es) * 1989-12-22 2012-10-29 Merck Serono Sa Constructos de ADN para la activación y la modificación de la expresión de genes endógenos
PT101031B (pt) * 1991-11-05 2002-07-31 Transkaryotic Therapies Inc Processo para o fornecimento de proteinas por terapia genetica

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7619007B2 (en) 2004-11-23 2009-11-17 Adamas Pharmaceuticals, Inc. Method and composition for administering an NMDA receptor antagonist to a subject
US8168209B2 (en) 2004-11-23 2012-05-01 Adamas Pharmaceuticals, Inc. Method and composition for administering an NMDA receptor antagonist to a subject
US8173708B2 (en) 2004-11-23 2012-05-08 Adamas Pharmaceuticals, Inc. Method and composition for administering an NMDA receptor antagonist to a subject

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US5981214A (en) 1999-11-09
CA2045175A1 (fr) 1991-05-07
EP0452484B2 (fr) 2004-07-28
DE69032809T2 (de) 1999-07-08
EP0945515A2 (fr) 1999-09-29
EP0945515A3 (fr) 2002-08-21
ES2090297T3 (es) 1996-10-16
KR100233781B1 (ko) 1999-12-01
AU7740791A (en) 1991-05-31
DE69027526D1 (de) 1996-07-25
ATE139574T1 (de) 1996-07-15
ATE174058T1 (de) 1998-12-15
DE69032809D1 (de) 1999-01-14
WO1991006667A1 (fr) 1991-05-16
NO912642D0 (no) 1991-07-05
SG122763A1 (en) 2006-06-29
CA2045175C (fr) 2003-03-18
NO309238B1 (no) 2001-01-02
GR3021105T3 (en) 1996-12-31
SG88714A1 (en) 2002-05-21
DE69027526T2 (de) 1996-12-05
AU635844B2 (en) 1993-04-01
US6015708A (en) 2000-01-18
GR3029328T3 (en) 1999-05-28
JPH04505104A (ja) 1992-09-10
WO1991006666A1 (fr) 1991-05-16
ES2127458T3 (es) 1999-04-16
EP0747485B1 (fr) 1998-12-02
JP2001186897A (ja) 2001-07-10
EP0747485A1 (fr) 1996-12-11
NO912642L (no) 1991-09-05
DK0452484T3 (da) 1996-10-14
ES2090297T5 (es) 2005-03-01
EP0452484A4 (en) 1992-05-13
DE69027526T3 (de) 2005-03-24
US5578461A (en) 1996-11-26
DK0747485T3 (da) 1999-08-16
EP0452484A1 (fr) 1991-10-23

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